Various diseases of the genitourinary tract can cause damage to the bladder and
result in urinary retention or incontinence. Treatment options of affected bladders
include augmentation cystoplasty, in which the bladder is enlarged by using a piece
of intestine. Since the use of intestine in the urinary tract can cause serious adverse
effects, new (bio)materials are being tested for bladder reconstruction. However, the
ideal (bio)material for reconstruction of the bladder has not yet been discovered,
mostly due to mechanical failure in vivo. A promising natural biomaterial is spider
silk, which is outstanding in its slow degradation rate and mechanical properties.
In this study, we assess the in vitro potential of spider silk as a suitable biomaterial
for bladder reconstruction by cultivating primary human urothelial cells (HUCs) on
native spider silk matrices. Here, we manufactured dental wire frames which were
woven with the dragline silk of female Nephila Clavipes spiders under standardized
conditions. Upon sterilization of spider silk frames, HUCs were cultivated on spider
silk matrices and evaluated for adhesion, expansion, viability and cellular differentiation.
Analysis of cell morphology and filamentous actin in HUCs revealed abundant
adhesion sites and bridging of HUCs to spider silk matrices. In addition, qRT-PCRbased
analysis of adhesion molecules present in HUCs revealed down-regulation
of certain adhesion molecules when HUCs were cultured on spider silk. Expansion
of HUCs on spider silk matrices was determined over time using Presto Blue and
DAPI cell count in fields of vision and showed that spider silk supports expansion of
HUCs. In vitro biocompatibility of spider silk was assessed by culturing HUCs in an
extract of spider silk and revealed no cytotoxic effects on HUCs. This is supported
by live/dead staining which demonstrated that HUCs were viable when cultured on
spider silk matrices. QRT-PCR-based analysis of epithelial-mesenchymal transition,
fibrosis and cellular differentiation markers expressed in HUCs when cultured on
spider silk showed, besides down-regulation of CD44, no differences compared to
control. Flow cytometry-based analysis of urothelial cellular differentiation markers
CD90, CD44 and CD49f demonstrated a small subpopulation lower in CD44 expression
at protein level when HUCs were cultured on spider silk. These results indicate
that spider silk causes HUCs to mature slightly faster, but has no major effects on
cellular differentiation of HUCs. Together, results describe a novel biomaterial which
supports adhesion, expansion and viability of HUCs. Our study gives new understanding
of cellular mechanisms occurring in HUCs when cultured on spider silk
and demonstrates that spider silk is a promising biomaterial for bladder reconstruction.